双酚A对男性生殖功能的影响及机制探讨

发布时间：2018-10-15 08:25

【摘要】：环境雌激素双酚A（Bisphenol A，BPA）是一种重要的化工原料，其广泛的应用范围导致了无处不在的暴露。BPA具有弱雌激素样作用和强抗雄激素样作用，可对雄性生殖系统产生影响。然而关于BPA雄性生殖毒性的人群研究和相关机制研究的报道极为有限。本研究通过人群研究探讨BPA对男性的生殖毒性，通过动物实验研究进一步探索可能的机制，为明确BPA的雄性生殖毒性及其机理提供科学依据。目的采用人群研究和动物实验研究相结合的方法，探讨BPA暴露对男性生殖功能的影响，并进一步探索可能的机制，为BPA对生殖功能的作用及其机制的研究提供新的思路。 1．通过分析BPA对男性精子质量、血清性激素水平以及性功能之间的关系，探讨BPA对男性的生殖毒性； 2．探讨BPA对雄性大鼠生殖系统及精子质量的影响，，以及对主要激素睾酮（T）与卵泡刺激素（FSH）的影响； 3．通过研究BPA对激素受体AR、FSHR和信号通路相关基因Src、ERK1/2、CREB的影响，初步探讨BPA在精子发生过程中可能的分子机制。方法人群研究：收集543名研究对象，采用结构式问卷调查研究对象一般人口学特征、既往病史及用药史、生活方式、性生活史、BPA暴露史等。采集研究对象尿液、精液、血液，应用高效液相色谱法(HPLC)检测尿液中BPA水平；计算机辅助精子分析系统检测精子数量、活动力和存活率，改良巴氏染色法检测精子形态学；放射免疫法(RIA)检测血清卵泡刺激素（FSH）、泌乳素（PRL）、睾酮（T）、雌二醇（E2）水平，酶联免疫吸附法(ELISA)检测血清抑制素B (INB)、游离睾酮（FT）、性激素结合球蛋白（SHBG）和雄烯二酮（AD）水平。应用SPSS16.0软件建立数据库并运用t检验、χ~2检验、趋势χ~2检验、pearson相关分析、spearman秩相关分析、多元线性回归分析等检验方法进行统计分析。动物实验：雄性Wistar大鼠（200±20g）28只按体重随机分为4组，每天固定时间灌胃染毒，染毒剂量分别为BPA0、50、100、200mg/（kg.d），连续4周后处死大鼠，采集生殖系统脏器、血清及一侧附睾尾精子。计算机辅助分析检测大鼠精子密度、活力、存活率，改良巴氏染色法检测精子畸形率；放射免疫法检测血清T、FSH水平。采用Real-time PCR和Western-blot法分别检测大鼠睾丸中AR、FSHR、Src、ERK1/2、CREB基因mRNA和蛋白表达水平。采用SPSS16.0建立数据库并运用单因素方差分析、Dunnet-t检验进行统计分析。结果人群研究 1．尿液中BPA浓度升高与精子密度低下、精子总数低下、精子活动力低下、精子存活率低下之间存在剂量效应关系；在调整可疑混杂因素后，尿液中BPA浓度与精子密度、总数、活动力、存活率之间存在负相关关系。 2．尿液BPA浓度升高与FT, AD, SHBG, PRL, FSH水平异常之间存在剂量效应关系。在调整可疑混杂因素后，尿液BPA浓度与AD、FSH水平之间存在负相关关系，与SHBG、PRL、E2水平之间存在正相关关系。 3．尿液中BPA浓度升高与男性性欲冲动程度、受刺激勃起能力、完成性交能力、射精强度、性生活满意度降低，以及勃起困难程度和射精困难程度升高之间存在剂量效应关系；在调整可疑混杂因素后，尿液中BPA浓度与各表现指标之间存在相关关系。动物实验 1．各染毒组体重增长量和染毒后体重之间存在差异，高剂量组体重增长量显著低于对照组；各染毒组大鼠精囊、前列腺、睾丸、附睾等性腺器官的脏器系数存在显著性差异，与对照组相比，中、高剂量组睾丸、精囊腺、前列腺脏器系数明显降低，高剂量组附睾脏器系数也明显降低。 2．各染毒组间精子质量存在显著性差异；与对照组相比，中、高剂量组精子活力和存活率显著降低；高剂量组精子密度也显著降低。 3．各染毒组间性激素T和FSH水平存在显著性差异；与对照组相比BPA中、高剂量组血清T水平明显降低，而高剂量组FSH水平明显升高；各染毒组间AR、FSHR mRNA和蛋白表达平均存在显著性差异；BPA中、高剂量组大鼠睾丸组织中AR、FSHR mRNA和蛋白水平均显著低于对照组。 4．未发现各染毒组间Src mRNA和蛋白以及蛋白磷酸化表达之间存在显著性差异；未发现各染毒组间ERK1/2mRNA和蛋白表达之间存在显著性差异,但ERK1/2蛋白磷酸化表达存在显著性差异，BPA中、高剂量组ERK1/2蛋白磷酸化水平显著低于对照组；未发现各染毒组间CREB mRNA和蛋白表达之间存在显著性差异,但CREB蛋白磷酸化表达存在显著性差异，BPA高剂量组CREB蛋白磷酸化水平显著低于对照组。结论 1．尿液BPA水平升高与男性精子质量下降、性激素水平异常、性功能下降相关； 2．高剂量BPA染毒对雄性Wistar大鼠有明显的生殖毒性，导致性腺器官受损，精子质量下降、激素水平改变； 3．BPA可导致大鼠睾丸组织中ERK1/2、CREB蛋白磷酸化水平降低，提示BPA可能通过对ERK1/2、CREB通路的抑制而影响生殖功能，但未发现Src与这一通路的上游调控有关。
[Abstract]:Environmental oestrogen double phenol A (BPA) is an important chemical raw material, and its wide application range leads to ubiquitous exposure. BPA has a weak estrogen-like action and a strong anti-androgen-like action, which can have an impact on the male reproductive system. However, there are very limited reports on population studies and related mechanism studies on BPA male reproductive toxicity. This study explored the reproductive toxicity of BPA to men through population studies, and further explored possible mechanisms through animal experiments to provide scientific basis for determining the male reproductive toxicity and its mechanism of BPA. Objective To explore the effect of BPA exposure on male reproductive function and explore possible mechanisms to study the role of BPA on reproductive function and its mechanism. To explore BPA's relationship between male sperm quality, serum sex hormone level and sexual function by analyzing BPA Reproductive toxicity to men; 2. Study BPA on male rats Effects of colonizing system and sperm quality, as well as on main hormone testosterone (T) and follicle The effect of BPA on hormone receptor AR, FSHR and signal pathway related genes Src, ERK1/ 2 and CREB was studied. Spermatozoa Possible molecular mechanisms in the course of the genesis: Study of population of 543 subjects, general demographic characteristics of subjects using structured questionnaires, past medical history, and use Drug history, lifestyle, history of sexual life, history of BPA exposure, etc. Collection of study subjects urine, semen, blood, application of high performance liquid chromatography (HPLC) to detect BPA levels in urine, computer-assisted sperm analysis system to detect sperm count, activity Serum follicle stimulating hormone (FSH), prolactin (PRL), testosterone (T), estradiol (E2) and enzyme linked immunosorbent assay (ELISA) were detected by radioimmunoassay (RIA). Serum Inhibin B (INB), Free testosterone (FT), Sex Hormone Binding The levels of globulin (SHBG) and androgradeone (AD) were established. The database was established with SPSS10.0 software and t test was used. Methods: Twenty-eight male Wistar rats (2000.20g) were randomly divided into 4 groups according to their weight, the daily fixed time was treated with daily dose of BPA0, 50, 100 and 200mg/ (kg 路 d). Detection of sperm density, viability, survival rate and improved pasteurization in rats by computer-assisted analysis of rat sperm density, viability, survival rate, and epididymal sperm. Serum T and FSH levels were measured by radioimmunoassay. Real-time PCR and Western-blot were used to detect AR, FSHR, Src, E in the testis of rats. RK1/ 2, CREB gene mRNA and protein expression level. Using SPSS10.0 to establish a database and use a single factor variance analysis Statistical analysis was conducted on Dunnet-t test. Results: 1. The increase of BPA concentration in urine was related to low sperm density, low sperm count, low sperm motility, and low sperm survival rate.; after adjusting the suspected confounding factors, urine There was a negative correlation between BPA concentration in liquid and sperm density, total number, living power and survival rate. There was a relationship between elevation and FT, AD, SHBG, PRL and FSH levels. After adjusting the suspected confounding factors, the urine BPA concentration was associated with AD, FSH. There is a negative correlation between the levels, and positive correlation exists between the levels of SHBG, PRL and E2. 3. The increase of BPA concentration in urine and the degree of male sexual desire, stimulated erection ability, completed sexual intercourse ability, ejaculation intensity, sex life satisfaction decreased, and there is a dose between the degree of erection difficulty and the degree of difficulty in ejaculation. effect relationship There was a correlation between BPA concentration in urine and each expression index after adjustment of suspicious confounding factors. Animal experiment 1. There was a difference between body weight growth amount and body weight after exposure, and the growth of body weight in high-dose group was significantly lower than that of control group. There was a significant difference in the organ coefficients of gonadal organs such as seminal vesicle, prostate, testis, epididymis and so on in each group. There was a significant decrease in the organ coefficient of the epididymis in the high-dose group compared with that in the middle and high-dose groups. The sperm quality among the groups was significantly lower than that in the high-dose group. Compared with the control group, the sperm motility and survival rate of the high-dose group decreased significantly; the sperm density of the high-dose group was also significantly decreased. Compared with the control group, the serum T level of the high-dose group was significantly lower than that in the control group. The levels of FSH in the high-dose group were significantly increased, and the expressions of AR, FSHR mRNA and protein in each group There was a significant difference between the levels of AR, FSHR mRNA and protein in the testis tissues of the rats with high dose group. The levels of AR, FSHR mRNA and protein in the testis tissues of the high dose group were significantly lower than those in the control group. There was a significant difference between ERK1/ 2mRNA and protein expression in the group, but there was a significant difference in the phosphorylation of ERK1/ 2 protein. In BPA, the phosphorylation level of ERK1/ 2 protein in high dose group was significantly lower than that in control group. exist Significant differences were noted, but there was a significant difference in the phosphorylation of CREB protein, and the BPA high dose group CREB The level of protein phosphorylation was significantly lower than that in control group. Conclusion 1. The level of BPA in urine and the quality of male sperm decreased, sex hormone level was abnormal, sex hormone level was abnormal, 2. High dose BPA exposure has obvious reproductive toxicity to male Wistar rats, resulting in impaired gonadal organs, decreased sperm quality, changes in hormone levels; 3. BPA can lead to ERK1/ 2, CR in rat testis tissues.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R114

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